The present invention relates to acellular pertussis vaccines, components thereof, and their preparation.
Whooping cough or pertussis is a severe, highly contagious upper respiratory tract infection caused by Bordetella pertussis. The World Health Organization estimates that there are 60 million cases of pertussis per year and 0.5 to 1 million associated deaths (ref. 1. Throughout this specification., various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately following the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure) In unvaccinated populations, a pertussis incidence rate as high as 80% has been observed in children under 5 years old (ref. 2). Although pertussis is generally considered to be a childhood disease, there is increasing evidence of clinical and asymptomatic disease in adolescents and adults (refs. 3, 4 and 5).
The introduction of whole-cell vaccines composed of chemically- and heat-inactivated B. pertussis organisms in the 1940""s was responsible for a dramatic reduction in the incidence of whooping cough caused by B. pertussis. The efficacy rates for whole-cell vaccines have been estimated at up to 95% depending on case definition (ref. 6) . While infection with B. pertussis confers life-long immunity, there is increasing evidence for waning protection after immunization with whole-cell vaccines (ref. 3). Several reports citing a relationship between whole-cell pertussis vaccination, reactogenicity and serious side-effects led to a decline in vaccine acceptance and consequent renewed epidemics (ref. 7). More recently defined component pertussis vaccines have been developed.
Various acellular pertussis vaccines have been developed and include the Bordetella pertussis antigens, Pertussis Toxin (PT), Filamentous haemagglutonin (FHA), the 69 kDa outer membrane protein (pertactin) and fimbrial agglutinogens (see Table 1 below. The Tables appear at the end of the specification).
Pertussis toxin is an exotoxin which is a member of the A/B family of bacterial toxins with ADP-ribosyltransferase activity (ref. 8). The A-moiety of these toxins exhibit the ADP-ribosyltransferase activity and the B portion mediates binding of the toxin to host cell receptors and the translocation of A to its site of action. PT also facilitates the adherence of B. pertussis to ciliated epithelial cells (ref. 9) and also plays a role in the invasion of macrophages by B. pertussis (ref. 10).
All acellular pertussis vaccines have included PT, which has been proposed as a major virulence factor and protective antigen (ref. 11, 12). Natural infection with B. pertussis generates both humoral and cell-mediated responses to PT (refs. 13 to 17). Infants have transplacentally-derived anti-PT antibodies (refs. 16, 18) and human colostrum containing anti-PT antibodies was effective in the passive protection of mice against aerosol infection (ref. 19). A cell-mediated immune (CMI) response to PT subunits has been demonstrated after immunization with an acellular vaccine (ref. 20) and a CMI response to PT was generated after whole-cell vaccination (ref. 13). Chemically-inactivated PT in whole-cell or component vaccines is protective in animal models and in humans (ref. 21) Furthermore, monoclonal antibodies specific for subunit S1 protect against B. pertussis infection (refs. 22 and 23).
The main pathophysiological effects of PT are due to its ADP-ribosyltransferase activity. PT catalyses the transfer of ADP-ribose from AND to the Gi guanine nucleotide-binding protein, thus disrupting the cellular adenylate cyclase regulatory system (ref. 24). PT also prevents the migration of macrophages and lymphocytes to sites of inflammation and interferes with the neutrophil-mediated phagocytosis and killing of bacteria (ref. 25). A number of in vitro and in vivo assays have been used to asses the enzymatic activity of S1 and/or PT, including the ADP-ribosylation of bovine transducin (ref. 26), the Chinese hamster ovary (CHO) cell clustering assay (ref. 27) , histamine sensitization (ref. 28), leukocytosis, and NAD glycohydrolase. When exposed to PT, CHO cells develop a characteristic clustered morphology. This phenomenon is dependent upon the binding of PT, and subsequent translocation and ADP-ribosyltransferase activity of S1 and thus the CHO cell clustering assay is widely used to test the integrity and toxicity of PT holotoxins.
Filamentous haemagglutonin is a large (220 kDa) non-toxic polypeptide which mediates attachment of B. pertussis to ciliated cells of the upper respiratory tract during bacterial colonization (refs. 9, 29). Natural infection induces anti-FHA antibodies and cell mediated immunity (refs. 13, 15, 17, 30 and 31). Anti-FHA antibodies are found in human colostrum and are also transmitted transplacentally (refs. 17, 18 and 19). Vaccination with whole-cell or acellular pertussis vaccines generates anti-FHA antibodies and acellular vaccines containing FHA also induce a CMI response to FHA (refs. 20, 32). FHA is a protective antigen in a mouse respiratory challenge model after active or passive immunization (refs. 33, 34). However, alone FHA does not protect in the mouse intracerebral challenge potency assay (ref. 28).
The 69 kDa protein is an outer membrane protein which was originally identified from B. bronchiseptica (ref. 35). It was shown to be a protective antigen against B. bronchiseptica and was subsequently identified in both B. pertussis and B. parapertussis. The 69 kDa protein binds directly to eukaryotic cells (ref. 36) and natural infection with B. pertussis induces an anti-P.69 humoral response (ref. 14) and P.69 also induces a cell-mediated immune response (ref. 17, 37, 38). Vaccination with whole-cell or acellular vaccines induces anti-P.69 antibodies (refs. 32, 39) and acellular vaccines induce P.69 CMI (ref. 39). Pertactin protects mice against aerosol challenge with B. pertussis (ref. 40) and in combination with FHA, protects in the intracerebral challenge test against B. pertussis (ref. 41). Passive transfer of polyclonal or monoclonal anti-P.69 antibodies also protects mice against aerosol challenge (ref. 42).
Serotypes of B. pertussis are defined by their agglutinating fimbriae. The WHO recommends that whole-cell vaccines include types 1, 2 and 3 agglutinogens (Aggs) since they are not cross-protective (ref. 43). Agg 1 is non-fimbrial and is found on all B. pertussis strains while the serotype 2 and 3 Aggs are fimbrial. Natural infection or immunization with whole-cell or acellular vaccines induces anti-Agg antibodies (refs. 15, 32). A specific cell-mediated immune response can be generated in mice by Agg 2 and Agg 3 after aerosol infection (ref. 17). Aggs 2 and 3 are protective in mice against respiratory challenge and human colostrum containing anti-agglutinogens will also protect in this assay (refs. 19, 44, 45).
The first acellular vaccine developed was the two-component PT+FHA vaccine (JNIH 6) of Sato et al. (ref. 46). This vaccine was prepared by co-purification of PT and FHA antigens from the culture supernatant of B. pertussis strain Tohama, followed by formalin toxoiding. Acellular vaccines from various manufacturers and of various compositions have been used successfully to immunize Japanese children against whopping cough since 1981 resulting in a dramatic decrease in incidence of disease (ref. 47). The JNIH 6 vaccine and a mono-component PT toxoid vaccine (JNIH 7) were tested in a large clinical trial in Sweden in 1986. Initial results indicated lower efficacy than the reported efficacy of a whole-cell vaccine, but follow-up studies have shown it to be more effective against milder disease diagnosed by serological methods (refs. 48, 49, 50, 51). However, there was evidence for reversion to toxicity of formalin-inactivated PT in these vaccines. These vaccines were also found to protect against disease rather than infection.
A number of new acellular pertussis vaccines are currently being assessed which include combinations of PT, FHA, P.69, and/or agglutinogens and these are listed in Table 1. Several techniques of chemical detoxication have been used for PT including inactivation with formalin (ref. 46), glutaraldehyde (ref. 52), hydrogen peroxide (ref. 53), and tetranitromethane (ref. 54).
Thus, current commercially-available acellular pertussis vaccines may not contain appropriate formulations of appropriate antigens in appropriate immunogenic forms to achieve a desired level of efficacy in a pertussis-susceptible human population.
It would be desirable to provide efficacious accellular pertussis vaccines containing selected relative amounts of selected antigens and methods of production thereof.
The present invention is directed towards acellular pertussis vaccine preparations, components thereof, methods of preparation of such vaccines and their components, and methods of use thereof.
In a further aspect of the invention, there is provided an immunogenic composition comprising the fimbrial agglutinogen preparation as provided herein. The immunogenic composition may be formulated as a vaccine for in vivo use for protecting a host immunized therewith from disease caused by Bordetella and may comprise at least one other Bordetella antigen. The at least one other Bordetella antigen may be filamentous haemagglutinin, the 69 kDa outer membrane protein adenylate cyclase, Bordetella lipooligosaccharide, outer membrane proteins and pertussis toxin or a toxoid thereof, including genetically detoxified analogs thereof.
In a further aspect of the invention, the immunogenic composition as provided herein may comprise at least one non-Bordetella immunogen. Such non-Bordetella immunogen may be diphtheria toxoid, tetanus toxoid, fcapsular pooysaccharide of Haemophilus, outer membrane protein of Haemoophilus, hepatitis B surface antigen, polio, mumps, measles and/or rubella.
The immunogenic compositions as provided herein may further comprise an adjuvant and such adjuvant may be aluminum phosphate, aluminum hydroxide, Quil A, QS21, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid or a lipoprotein.
In accordance with one aspect of the present invention, there is provided a vaccine composition for protecting an at-risk human population against a case of disease caused by infection by B. pertussis, which comprises pertussis toxoid, filamentous haemagglutinin, pertactin and agglutinogens in purified form in selected relative amounts to confer protection to the extent of at least about 70% of members of the at-risk population.
Such vaccine composition may contain about 5 to about 30 xcexcg nitrogen of pertussis toxoid, about 5 to about 30 xcexcg nitrogen of filamentous haemagglutinin, about 3 to about 15 xcexcg nitrogen of pertactin and about 1 to about 10 xcexcg nitrogen of agglutinogens.
In one specific embodiment, the vaccine may comprise pertussis toxoid, filamentous haemagglutinin, the 69 kDa protein and filamentous agglutinogens of Bordetella at a weight ratio of about 10:5:5:3 as provided by about 10 xcexcg of pertussis toxoid, about 5 xcexcg of filamentous haemagglutinin, about 5 xcexcg of 69 kDa protein and about 3 xcexcg of fimbrial agglutinogens in a single human dose. In a further particular embodiment, the vaccine may comprise pertussis toxoid, filamentous haemagglutinin, 69 kDa protein and fimbrial agglutinogens in a weight ratio of about 20:20:5:3 as provided by about 20 xcexcg of pertussis toxoid, about 20 xcexcg of filamentous haemagglutinin, about 5 xcexcg of 69 kDa protein and about 3 xcexcg of fimbrial agglutinogens in a single human dose. In a yet further particular embodiment, the vaccine may comprise pertussis toxoid filamentous haemagglutinin, 69 kDa protein and fimbrial agglutinoaens in a weight ratio of about 20:10:10:6 as provided by about 20 xcexcg of pertussis toxoid, about 10 xcexcg of filamentous haemagglutinin, about 10 xcexcg of 69 kDa protein and about 6 xcexcg of fimbrial agglutinogens in a single human dose.
The extent of protection to the at-risk human population afforded by the vaccine composition of the invention may be at least about 80%, preferably about 85%, for a case of spasmodic cough of duration at least 21 days and culture-confirmed bacterial infection. The extent of protection to the at-risk human population may be at least about 70% for a case of mild pertussis having a cough of at least one day duration.
The agglutinogens component of the vaccine preferably comprise fimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen 3 (Agg 3) substantially free from agglutinogen 1. The weight ratio of Agg 2 to Agg 3 may be from about 1.5:1 to about 2:1.
The vaccine provided herein may be combined with tetanus toxoid and diphtheria toxoid to provide a DTP vaccine. In one embodiment, the vaccine contains about 15 Lfs of diphtheria toxoid and about 5 Lfs of tetanus toxoid.
In addition, the vaccine may also comprise an adjuvant, particularly alum.
In a further aspect of the present invention, there is provided a method of immunizing an at-risk human population against disease caused by infection by B. pertussis, which comprises administering to members of the at-risk human population an immunoeffective amount of the vaccine composition provided herein to confer protection to the extent of at least about 70% of the members of the at-risk population.
Advantages of the present invention include an improved acellular pertussis vaccine composition of increased efficacy.
The present invention further provides, in an additional aspect, purified forms of pertussis toxin, filamentous haemagglutinin, pertactin and fimbrial agglutinogens of B. pertussis when used in the manufacture of a vaccine composition for administration to an at-risk human population to confer protection to the extent of at least about 70% of the members of said at-risk human population.
In such use, there may be employed in the manufacture of a single human dose of the vaccine composition from about 30 xcexcg of nitrogen of pertactin and about 1 to about 10 xcexcg of nitrogen of the fimbrial agglutinogens. In particular, the vaccine composition as provided herein have been selected by the National Institute of Allergy and Infectious Diseases (NIAID) of the United States Government for evaluation in a double-blind, human efficacy clinical trial, thereby establishing a sufficient basis to those especially skilled in the art that the compositions will be effective to some degree in preventing the stated disease (pertussis). The subject of that trial (being a vaccine as provided herein) has met the burden of being reasonably predictive of utility.